Mass spectrometry is an extraordinarily powerful bioanalytical technique that has had a profound impact on our molecular understanding of human health and disease. Major advances in mass analyzer technology, dissociation techniques, and ionization methods are largely attributed to the central role that mass spectrometry plays in the field of systems biology. While mass spectrometry has evolved over the last century into a highly effective analytical tool, there are still opportunities for new advances to be made allowing an even more diverse array of biological questions to be addressed. This proposal is centered on the development of new ionization methods for biological mass spectrometry to allow for tissue imaging across several classes of biological molecules. The short-term objective of this proposal is to further develop and fundamentally understand this innovative ionization method using real biological systems. These results will provide a solid foundation from which biological applications will directly benefit. In this mindse, we will develop and apply these new ionization methods to tissue imaging. The long-term objective is to establish these new ionization methods as an enabling bioanalytical technology to effectively address questions in human health and disease. Mass spectrometry (MS), the science related to the weighing of molecules, has had a profound impact on the study of human health and disease including cancer, heart disease, neural development, and auto-immune diseases. A prerequisite of MS is to convert neutral molecules into charged species (ions) such that they can be weighed by the mass spectrometer. The focus of this research is to develop new ionization methods allowing a more diverse array of contemporary biomedical questions to be addressed. This will include the imaging of tissues to ultimately provide new biological insights.